Transgenerational influence of parental morphine exposure on pain perception, anxiety-like behavior and passive avoidance memory among male and female offspring of Wistar rats

Accumulating evidence suggests that epigenetic mechanisms play an important role in the formation and maintenance of memory within the brain. Moreover, the effect of parental drug-exposure before gestation on behavioral state of offspring has been little studied. The main objective of the current study is to evaluate the effect of parental morphine exposure on avoidance memory, morphine preference and anxiety-like behavior of offspring. The total of 32 males and 32 females were used for mating. The animals were treated with morphine. The offspring according to their parental morphine treatment was divided into four groups (n=16) including paternally treated, maternally treated, both of parents treated and naïve animals. The pain perception, anxiety-like behavior, and avoidance memory were evaluated in the offspring. In the current study, the total of 256 offspring was used for the experiments (4 tasks × 4 groups of offspring × 8 female offspring × 8 male offspring). The finding revealed that the avoidance memory and visceral pain were reduced significantly in male and female offspring with at least one morphine-treated parent. Moreover, anxiety-like behavior was reduced significantly in the male offspring with at least one morphine-treated parent. While anxiety-like behavior was increased significantly in female offspring that were treated by morphine either maternally or both of parents. The data revealed that the endogenous opioid system may be altered in the offspring of morphine-treated parent(s), and epigenetic role could be important. However, analysis of variance signified the important role of maternal inheritance.

The total of 32 males and 32 females were used for mating. The animals were treated with morphine. The offspring according to their parental morphine treatment was divided into four groups (n=16) including paternally treated, maternally treated, both of parents treated and naïve animals. The pain perception, depressive-like behavior, and avoidance memory were evaluated in the offspring. In the current study, the total of 256 offspring was used for the experiments (4 tasks × 4 groups of offspring × 8 female offspring × 8 male offspring

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Value of the data  The data would be useful to assess effect of parental morphine exposure on the behavior of the offspring  The comparison between female and male offspring that parentally were exposed to addictive drugs rarely has been discussed.
 The data would be useful for the researchers interested in epigenetic or transgenerational influence of parental morphine exposure on the child  The data could be useful for researchers who are interested on the long-term effect of opioids exposure and other addictive substances on the individuals.

Animals
All experimental procedures were in agreement with the regulations of the experimental animal ethics at Tehran University of Medical Sciences ethics committee. Wistar albino rats, weighing between 200 to 220 gram were purchased from Pasture Institute, Tehran, Iran. The rats were exposed to morphine according to a modified protocol of Akbarabadi and colleagues (Akbarabadi et al., 2018). The total of 32 males and 32 females were used for mating and morphine treatment. The total of 128 male and 128 female offspring were used for the experiments (4 tasks × 4 groups of offspring × 8 female × 8 male). The animals were maintained in Plexiglas cages (n=4) with free access to fresh water and food at constant temperature 22±2 ºC and light/dark cycle (07:00-19:00 h). The biological father was removed before the birth of the offspring and the biological mother was kept until the end of the breastfeeding period. The experiments were started when the offspring were reached to 8 weeks old. Moreover, female rats were tested on the diestrous phase of the estrous cycle and vaginal smear test was monitored daily. Moreover, to avoid potential confounding factory the offspring were no over-represented in the experiments. Furthermore one week before starting of the experiment the animals were picked up daily to reduce handling anxiety (Gouveia and Hurst, 2013).

Parental morphine exposure
Twenty-four male and twenty-four female Wistar rats were exposed to the treatment of oral morphine sulfate according to the protocol described earlier (Akbarabadi et al., 2018). Morphine was given in the drinking fluid in the range of 0.1 to 0.4 mg.ml -1 in 48 intervals for up to three weeks ( Figure 1A). Sucrose (2 %) was added to diminish the bitter taste of morphine.
Eight male and eight female rats were considered as the control group which only received sucrose (2 %). Naloxone was administrated intraperitoneally (IP) for all of the animals to confirm the morphine dependence. Withdrawal symptoms and the average of morphine consumption were recorded ( Figure 1B and C).

Mating protocol
Ten days after the last morphine administration, the animals were assigned for mating. The offspring of the animals was arranged in four groups as offspring of healthy parents (naive), offspring of morphine-treated female and healthy male rats (maternally treated), offspring of morphine-treated male and healthy female rats (paternally treated) and offspring of morphinetreated male and female rats (both of parents were treated).

Passive-avoidance memory test
The passive avoidance memory is evaluated according to the protocol that is described earlier (Akbarabadi et al., 2018). The avoidance memory apparatus consists of a box with two compartments that were separated by a guillotine door. In the learning trial, the animals were placed in the light compartment and were allowed to cross the dark compartment, the guillotine door was closed and received an electric shock via gird floor (50 Hz, 1 mA, and 5s). The latency time of each animal to cross to the dark compartment was recorded. The experiment was repeated and in case of no entrance to the dark compartment within 120s, a successful acquisition of avoidance memory was recorded. The animals with successful acquisition were subjected to the test trial. In the test trial, each animal was placed in the light compartment and the door was opened. The step-through latency for crossing to the dark compartment was recorded for each animal. The testing trial ended either when the animal entered into the dark compartment or remained in the light compartment until the cut-off time (300 s).

Open-field test
The open-field test is a behavioral test to evaluate depressive-like behavior based on the locomotor activity (Damián et al., 2014;Motaghinejad et al., 2016). The open-field apparatus consists of a plexiglass square box with walls to reduce outside noise and light. Each rat was placed at the center of the apparatus and left to move freely for 10 min. The number of times that the animal preened its fur or tail with its mouth or forepaws (grooming), square crossed (locomotion) and the number of times that a rat reared up on its hind limbs (rearing) were recorded. After each monitor, the cage was cleaned with 70 % ethanol solution and left to dry.

Forced swimming test
The rats that were used for the open-field test were re-used in the forced swimming test. Forced swimming test is conducted to assess depressive-like behavior acording to the protocol described earlier (Porsolt et al., 1977). Briefly, the rat was placed on a Plexiglas cylinder (60 × 30 cm) that was filled with water (25 °C) to the height of 30 cm. In trial day the rats were habituated to the environment by swimming in the cylinder for 5 min in 24 h before test day. In the test day each rat was allowed to swim for 5 min while a video camera recorded from above. The latency to immobility and the total time of immobility were measured as an index of depressive-like behavior.

Writhing test
The writhing test is a method to evaluate nociception. Each animal was placed on a small observation chamber. After 10 min of habituation a volume of 10 ml.kg -1 acetic acid (0.8 %) was i.p administrated. The nociceptive behavior characterized by abdominal contraction known S5 as writhing, is described as an exaggerated extension into the abdomen combined with the outstretching of hind limbs. Five minutes following the administration, the number of writhing and total time of writhing was recorded over 10 minutes (Singh et al., 1983).

Formalin test
The formalin test was performed to evaluate acute and chronic pain. The pain is induced by applying 0.1 ml of 2.5 % formalin (Merck, Germany) into the dorsal surface of the left hind paw of each rat, and the rats were placed in an observation chamber with a mirror mounted on three sides to allow a clear view of the paws. The total times that each rat spent licking the injected paws were recorded. Acute pain as a result of nociceptor stimulation was observed in 1-10 min interval and the persistent pain was observed in 20-40 min interval of formalin injection (Hunskaar et al., 1985).

Statistical analysis
The chance of false positive results was reduced by normality analysis using Kolmogorov-Smirnov (K-S) test. The normal data with K-S value more than 0.01, were subjected to twoway ANOVA analysis followed by Dunnett's post-hoc mean comparison test. The p-values lower than 0.05 were considered significant. All statistical analyses were conducted using IBM SPSS 21 software. The output of the statistical analysis is summarized in Table 2. S6 Data:  The table shows the Fisher's values (F value) for each dependent variable. The values were calculated using IBM SPSS software. The significant difference at 0.05 level (P value < 0.05) was represented with one star (*). The significant difference at 0.01 level (P value < 0.01) was represented with two stars (**).